CN103122315B - Antrodiella zonata and bacterial reagent made from same for degrading metalaxyl pesticide residue - Google Patents
Antrodiella zonata and bacterial reagent made from same for degrading metalaxyl pesticide residue Download PDFInfo
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- CN103122315B CN103122315B CN201210506078.1A CN201210506078A CN103122315B CN 103122315 B CN103122315 B CN 103122315B CN 201210506078 A CN201210506078 A CN 201210506078A CN 103122315 B CN103122315 B CN 103122315B
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Abstract
The invention provides antrodiella zonata and a bacterial reagent made from antrodiella zonata for degrading metalaxyl pesticide residue. The strain is a white corroded fungus and is identified as antrodiella zonata D7821 which belongs to bracket fungus, Basidiomycetes. The strain is collected and separated from broad-leaved tree rottenwood of a primeval forest, and the strain has the main cultivating characteristic that the bacterial colony is loose, flocculent and white. ITS sequence of the strain is submitted to GenBank, with the register number of KC218571. Degrading bacteria cultivated by liquid and fermented by straw in solid state are applied to soil of vegetable fields in protected land polluted by metalaxyl pesticide, and the pesticide residue in the soil within 18 days can be reduced by over 75%. As the bacterial reagent for degrading metalaxyl pesticide residue, produced according to the technical scheme can effectively repair soil of the vegetable fields in the protected land polluted by pesticide, the toxicity of pesticide residue to a human body is reduced, and the ecological environment is protected.
Description
Technical field
The invention belongs to environmental microorganism field; to utilize the chemical pesticide in method of microorganism degraded protection ground horticultural vegetable field soil residual, relate to a strain separation on the rotten wood of virgin forest to oomycetes sterilant---metaxanin has the little Antrodia bacterial strain of the endless belt of efficient degradation effect (Antrodiella zonata).
Technical background
Along with agricultural chemicals is produced in a large number and is widely used, when it brings the huge agricultural income of people, also caused the severe contamination of environment, chemical pesticide is also subject to people's attention day by day to the serious threat of human health.Be applied to the agricultural chemicals on crop, wherein a part is attached on crop, and a part is scattered in the environment such as soil, large G&W, and the part in the remaining agricultural chemicals of environment can be absorbed by plants again.Remains of pesticide directly passes through fruit or water, atmosphere arrives in people, animal body, or finally passes to people, animal by environment, food chain, causes the long-term hazards to human body.In order to adapt to the heavy demand of market to vegetables in improper season fruit, protection ground (booth and greenhouse) area increases year by year.Due to planting vegetable and fruit year after year; high frequency is used protection ground soil to cause soil fertility decline; in enclosed space, all kinds of phytopathogens grow in a large number; in order to suppress these pathogenic bacterias, must use each series bactericidal agent of greater concn, so just cause the residual lasting accumulation of chemical pesticide in soil.Therefore explore the method that pesticide residue are carried out effectively degrading and become problem anxious to be resolved.In this area research, the use microorganism of extensively rising in recent years carries out biological degradation to pesticide residue becomes hot fields, by microbial process, agricultural chemicals macromole is resolved into micromolecular compound, make it lose the harm to human body, and can not cause secondary pollution to environment, so microorganism has great potential to degrading pesticide residues.White-rot fungi is that a class can be grown on wood substrates, cause a class fungi of timber white rot, except xylogen, Mierocrystalline cellulose and the hemicellulose that can degrade in timber, the multiple environmental pollutant of can also effectively degrading, the synthetic chemical pesticide that comprises current many types, the enzyme systems such as the laccase of secreting in its process of growth, manganese peroxidase and lignin peroxidase can these environmental pollutant of non-specific degraded.White-rot fungi is of a great variety; its degradation capability to environmental pollutants also varies; therefore obtain the efficient degrading bacterial strain of chemical pesticide and use the pesticide residual contamination in specific method of application degraded soil; to effective restoration and protection ground field of vegetables pesticide contaminated soil, reduce pesticide residue and poison, preserve the ecological environment significant to human body.
This patent is the high-efficiency degradation oomycetes sterilant that separation obtains from the corruption wood of virgin forest---the white-rot fungi strains A ntrodiella zonata of metaxanin, experiment shows that this bacterium can utilize metaxanin and other several oomycetes bactericide agricultural chemicals to grow as sole carbon source and the energy in water agar, and makes its degraded; In use, take rice straw after substratum carries out solid state fermentation, culture is added into and remains metaxanin, this bacterium can more than 75%, have good degradation effect by the metaxanin degraded in soil in 18 days.Absolutely prove that this bacterium can be for the protection of the biological degradation of ground horticultural vegetable field soil metaxanin pesticide residue.There is no at present the bibliographical information that utilizes the research of the little Antrodia degraded of endless belt metaxanin pesticide residue.
Summary of the invention
The object of this invention is to provide a kind of separation from rotten wood can efficient degradation oomycetes sterilant---the little Antrodia bacterial strain of endless belt of metaxanin, the method for utilizing this bacterial strain to degrade to the metaxanin pesticide residue in protection ground horticultural vegetable field soil.
One strain metaxanin pesticide degradation bacteria, this microorganism is a kind of white rot fungi, through being accredited as the little Antrodia of endless belt (Antrodiella zonata), the little Antrodia bacterial strain of this endless belt is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation date: on November 15th, 2012, deposit number was: CGMCC6810.Bacterium colony is flocculence, oyster white; Bacterium colony is comprised of generative hyphae, and generative hyphae thin-walled arrives slightly heavy wall, typically has a diameter from 1.8-3 micron, occasionally be expanded to 6 microns, clamp connexion is separated, common bilateral clamp connexion, normal branch, mycelia content is had a liking for blue reaction without having in the blue reagent of cotton, unchanged in potassium iodide reagents; Plate is cultivated 3 days colony diameter 3-4 centimetre;
Can be saprophytic in multiple deciduous tree, to fall wood upper, decompose and utilize xylogen, Mierocrystalline cellulose and the hemicellulose in timber, and can take metaxanin agricultural chemicals and grow as unique nutrition source.The GenBank(accession number of the ITS sequence of this bacterial strain is KC218571).
The microbial inoculum that described metaxanin pesticide residual degrading bacteria is produced, production stage is:
(1) actication of culture: described in picking, the little Antrodia inoculation of endless belt, in being added with the 60mm culture dish of separating and preserving substratum, is cultivated 4-5 days at 28-30 ℃;
(2) above-mentioned cultured bacterial classification is broken into the mycelia small pieces of 6mm, inoculate in 500ml Erlenmeyer flask liquid nutrient medium, under 28-30 ℃, 140-160rmp/min rotating speed, 4-5 days is cultivated in concussion;
(3) by the cultured fermented liquid of above-mentioned steps together with mycelium pellet, with interior cut refiner, in 5000rmp/min, carry out homogenate 3 times, each homogenate time 15-20 second;
(4), by straw segment, segment length 5mm left and right, adds distilled water to make straw section water content 50-60%(W/W), then through 121 ℃ of high-temperature sterilization 20-30 minute;
(5) get the good mycelium inoculation liquid of homogenate in step (3) straw section is inoculated, inoculum size 10-15%(W/W); After inoculation, mix, insert in length and width high 40cm * 25cm * 5cm fermentation dish straw section cumulative volume 3.5-4L;
(6) straw section inoculum is placed in to incubator and carries out solid-state aerobic fermentation, sterile air air flow 0.8-1(V/Vmin), culture temperature 30-32 ℃; Cultivate solid state fermentation after 15-18 days and complete, take out fermented product and stir.
Liquid culture based formulas is: Fructus Hordei Germinatus soaks powder 10g, potassium primary phosphate 0.5g, CuSO
40.1g, CaCl
20.01g, MnSO
40.01g, pH nature, adds water and is settled to 1L.
Strain separating storage medium: Fructus Hordei Germinatus soaks powder 10g, glucose 10g, agar 18g, adds water to 1L, pH value nature.
Advantage of the present invention is: the little Antrodia of this endless belt is that the strain obtaining through Large-scale Screening has higher vigor, the efficient degrading bacterial strain very capable to metaxanin chemical residual degradation, its cultural method is simple, fast growth, be difficult for variation, plurality of enzymes that it produces in process of growth system is can non-specific degraded Multiple Pesticides residual, can be used as the type strain of research white-rot fungi to metaxanin chemical residual degradation mechanism; Also can use its rice straw fermented culture of preparing to degrade to the pesticide residue in protection ground horticultural vegetable field soil.This bacterial strain has the metaxanin pesticide residue of water body and soil is carried out to biodegradable industrial applications prospect; To effective restoration and protection ground field of vegetables pesticide contaminated soil, reduce pesticide residue to human body poison, preserving the ecological environment has important value.
Embodiment
The separation from the rotten wood of virgin forest of the bacterial strain of degraded metaxanin pesticide residue provided by the present invention obtains.
This bacterium separation and cultural method are as follows:
1. substratum preparation:
(1) strain separating storage medium (solid, 1L): Fructus Hordei Germinatus soaks powder 10g, glucose 10g, agar 18g, adds water to 1L, pH value nature;
(2) liquid nutrient medium (liquid, 1L): Fructus Hordei Germinatus soaks powder 10g, potassium primary phosphate 0.5g, CuSO
40.1g, CaCl
20.01g, MnSO
40.01g, pH nature, adds water and is settled to 1L.
(3) straw substratum (solid, dry rice straw 1Kg): cutted straw (5mm is long) 1Kg, adds water 1500g.Above substratum is 121 ℃ of sterilizing 20-30 minute in pressure kettle all.
2. actication of culture, liquid culture and inoculation liquid preparation:
Actication of culture: described in picking, the little Antrodia inoculation of endless belt, in being added with the 60mm culture dish of separating and preserving substratum, is cultivated 4-5 days at 28-30 ℃;
With scalpel scraping media surface mycelia, connect bacterium and enter in liquid nutrient medium, in 28-30 ℃, shaking culture 4-5 days under 140-160rmp/min speed conditions, obtains the nutrient solution that comprises a large amount of mycelium pellets.Above-mentioned cultured fermented liquid, together with mycelium pellet, is carried out to homogenate 3 times with interior cut refiner in 5000rmp/min, each homogenate time 15-20 second.
3. the preparation of straw section culture: by straw segment, segment length 5mm left and right, adds distilled water to make straw section water content 50-60%(W/W), then through 121 ℃ of high-temperature sterilization 20-30 minute.
4. the inoculation of straw section and solid ventilation fermentation: the mycelium inoculation liquid that homogenate is good is inoculated straw section, inoculum size 10-15%(W/W); After inoculation, mix, insert in length and width high 40cm * 25cm * 5cm fermentation dish straw section cumulative volume 3.5-4L.Straw section inoculum is placed in to incubator and carries out solid-state aerobic fermentation, sterile air air flow 0.8-1(V/Vmin), culture temperature 30-32 ℃; Cultivate solid state fermentation after 15-18 days and complete, take out fermented product and stir.
The microbial inoculum of being produced by technical solution of the present invention can effectively be degraded and be protected metaxanin pesticide residue in ground horticultural vegetable field soil.
Embodiment 1
The cultivation of bacterial classification: in the plate of above-mentioned strain separating storage medium, be in 30 ℃ incubators to cultivate 4 day in temperature with the mycelium inoculation of the little Antrodia Antrodiella of inoculating needle picking endless belt zonata, bacterium colony is loose, snowy white, flocculence.With the loose mycelia of scalpel scraping, be inoculated in the Erlenmeyer flask that 500ml liquid nutrient medium of autoclaving 20min under 121 ℃, 0.1MPa is housed.Be placed in again 30 ℃, rotating speed 140rmp/min, shaking table concussion is cultivated 5 days, obtains mycelium pellet and nutrient solution.
Straw solid state fermentation: dry rice straw chopping, the about 5mm of straw section is long, gets 1kg and inserts PE box with cover, adds water 600g, stirs, in 121 ℃ of sterilizing 20-30 minute.Taking-up, through the cultured bacterial classification of aforesaid liquid, is carried out homogenate with interior cut refiner, rotating speed 5000rmp/min, homogenate 30 seconds.Get mycelia suspension inoculation straw substratum, inoculum size inoculum size 15%(W/W), in 30 ℃, aerated culture 18 days, air flow 0.8(V/Vmin).
Embodiment 2 degradeds of solid state fermentation degradation bacterium preparation to metaxanin pesticide residue
(1) get the protection ground horticultural vegetable field soil 3kg that metaxanin content is 5mg/kg, soil moisture content 60%, crossing aperture is 1mm
2nylon mesh screen is removed bulk stone and foreign material;
(2) get the about 1.5kg of straw section solid state fermentation degradation bacterium preparation, add soil, and stirring and evenly mixing, by homomixture tiling, thickness 5cm, cultivates 18 days in 30 ℃.2 days during this time atomized water sprays 1 time are to maintain the humidity of homomixture 60% left and right.With the straw without inoculation, the soil of the metaxanin agricultural chemicals that contains equal concentrations, the two mixes as a control group.
(3) every sampling in 3 days once, get 3g pedotheque and be placed in 50mL plastic centrifuge tube, add acetonitrile 10mL, the 30min that vibrates on vibrator fully extracts (model: QL-861 and frequency 2400rmp/min).Suspension is carried out to centrifugal (model: KDC-40), revolution 4000rmp/min, takes out supernatant liquor.
(4) repeating step is (3) 3 times, merge supernatant liquor, in solid-phase extraction device through Fo Luoli tripoli solid-phase extraction column (specification: 500mg3ml) purify, Rotary Evaporators concentrated (40 ℃ following), lyophilize at-20 ℃, with a small amount of acetonitrile, dissolve the pesticide residue of extracting again, it is carried out to gas chromatographic analysis.Calculate degradation rate: degradation rate (%)=(A-B)/A * 100, wherein A is control group metaxanin pesticide residue value (after 18 days, residual value is 4.1mg/L), B is the metaxanin pesticide residue value (after 18 days, residual value is 1mg/L) after degradation bacterial agent degraded.Under this culture condition, the little Antrodia Antrodiella of endless belt zonata can reach more than 75% the degradation rate of metaxanin pesticide residue for 18 days.
Claims (3)
1. the little Antrodia of endless belt, it is characterized in that: this microorganism is a kind of white rot fungi, through being accredited as the little Antrodia of endless belt (Antrodiella zonata), the little Antrodia bacterial strain of this endless belt is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on November 15th, 2012, deposit number is: CGMCC6810.
2. the microbial inoculum that the little Antrodia of endless belt claimed in claim 1 is produced, production stage is:
(1) actication of culture: the little Antrodia inoculation of picking endless belt claimed in claim 1, in being added with the 60mm culture dish of separating and preserving substratum, is cultivated 4-5 days at 28-30 ℃; Strain separating storage medium is: Fructus Hordei Germinatus soaks powder 10g, glucose 10g, and agar 18g, adds water to 1L, pH value nature;
(2) above-mentioned cultured bacterial classification is broken into the mycelia small pieces of 6mm, inoculate in 500ml Erlenmeyer flask liquid nutrient medium, under 28-30 ℃, 140-160rpm rotating speed, 4-5 days is cultivated in concussion;
(3) by the cultured fermented liquid of above-mentioned steps together with mycelium pellet, with interior cut refiner, in 5000rpm, carry out homogenate 3 times, each homogenate time 15-20 second;
(4) by straw segment, segment length 5mm left and right, adding distilled water to make straw section moisture content is 50-60%, then through 121 ℃ of high-temperature sterilization 20-30 minute;
(5) get the good mycelium inoculation liquid of homogenate in step (3) straw section is inoculated, inoculum size weight percent is 10-15%; After inoculation, mix, insert in length and width high 40cm * 25cm * 5cm fermentation dish straw section cumulative volume 3.5-4L;
(6) straw section inoculum is placed in to incubator and carries out solid-state aerobic fermentation, the volume ratio of sterile air air flow per minute ventilation is 0.8-1, culture temperature 30-32 ℃; Cultivate solid state fermentation after 15-18 days and complete, take out fermented product and stir all.
3. press the microbial inoculum that the little Antrodia of endless belt described in claims 2 is produced, liquid culture based formulas is: Fructus Hordei Germinatus soaks powder 10g, potassium primary phosphate 0.5g, CuSO
40.1g, CaCl
20.01g, MnSO
40.01g, pH nature, adds water and is settled to 1L.
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